蒙古沙冬青三个寒旱诱导表达NAC转录因子基因的功能研究

NAC transcription factors have been identified to play important roles in plant resistance to adverse environments. Many NACs have been identified significantly enhanced the resistance of their transgenic plants to cold, drought and other stresses by inducing the expression of many stress-related genes. Ammopiptanthus mongolicus is the unique evergreen broadleaf plant endemic mainly to the west desert of Inner Mongolia and has strong resistance to harsh environments, especially to cold and drought. In our previous study, we have identified three cold- and drought-induced NAC genes by transcriptome sequencing and expression profiling of this species. We have cloned the complete coding region cDNAs of these AmNACs and successfully inserted them into plant expression vectors. Overexpression of AmNAC1, one of the three AmNACs, in Arabidopsis seedlings improved their freezing resistance. In the present study, we will perform in-depth functional analyses of these genes. We aim at:(1) to ascertain their expression profiles in response to different abiotic stresses and different phytohormons such as ABA, and their expression levels in different organs; (2) to determine their subcellular localization and their transcriptional activation activity; (3) to characterize their functions in resistance to cold, drought, salt and heat stresses and to analyze their regulatory mechanisms by using their transgenic plants. We believe that those results will contribute a great deal to our understanding of the resistant mechanisms of A. mongolicus and even other plants to harsh environments, especially to cold and drought. The project also is expected to provide useful genes for improving plant stress resistance by genetic engineering.

NACs是近年来发现的一类重要植物抗逆转录因子，可通过诱导许多抗逆相关基因的表达而提高植物对干旱和低温等逆境的抗性。蒙古沙冬青是内蒙古西部荒漠区唯一的常绿阔叶植物，具有很强的抗寒抗旱等抗逆特性。我们在前期通过转录组测序和表达谱分析，从该植物获得三个受寒旱诱导的NAC基因，并完成其编码区cNDA的克隆和植物表达载体构建。将其中AmNAC1在拟南芥中超表达，初步证明可提高其抗冻性。本项目拟在前期研究基础上，对三个AmNAC基因进行深入的功能分析，以明确其响应不同非生物胁迫和ABA等激素的表达模式，以及在不同器官中的表达水平；确定其亚细胞定位和转录激活活性；探知其在转基因拟南芥抵抗寒旱盐热等逆境中的功能，解析其抗逆作用机理。研究结果不仅为揭示蒙古沙冬青乃至其他植物抗寒抗旱等分子机理提供新知识，同时可望为植物抗逆基因工程提供新的基因资源。

NAC类转录因子是植物中一类重要的抗逆相关调节蛋白，可通过诱导许多抗逆功能基因的表达而提高植物的抗逆性。从强抗逆植物中克隆和研究NAC基因具有重要意义。沙冬青是中亚荒漠区唯一的常绿阔叶植物，具有很强的抗寒和抗旱等抗逆特性。本项目首次从蒙古沙冬青中克隆到6个NAC基因（AmNAC1~AmNAC6），对其基本结构、表达模式、编码蛋白的亚细胞定位和转录激活活性，以及在植物抗逆性中的作用和调节机理进行了研究，主要结果如下：.1. AmNAC1~AmNAC5的gDNA编码区内均含有2个内含子、3个外显子。.2. AmNAC1~AmNAC5均参与沙冬青对不同逆境胁迫的响应，而且可能与ABA等激素信号途径有关；AmNAC3~AmNAC5、尤其AmNAC4在沙冬青响应秋末和冬季寒冷天气中起重要作用；AmNAC6对多种逆境胁迫无明显响应。此外，AmNAC1主要在侧根、花和未成熟果荚中表达；AmNAC2主要在侧根中表达；AmNAC3在侧根和嫩枝中表达量较高；AmNAC4主要在嫩叶中表达，其次是嫩枝和侧根；AmNAC5主要在侧根和嫩叶中表达；AmNAC6在嫩叶中表达量较低，在其他器官中表达量较高。.3. AmNAC1~AmNAC6的编码蛋白均定位于细胞核内，其中AmNAC1和AmNAC3~AmNAC5均具有转录激活活性，而且转录激活区均在其C端。.4. 对转基因拟南芥的抗逆性鉴定结果表明：AmNAC1对增强抗旱性和抗冻性有较明显作用；AmNAC2对抵抗各种非生物胁迫作用较弱；AmNAC3可以显著提高耐盐性，但同时降低了耐热性；AmNAC4在抵抗干旱、低温和盐胁迫中均有显著作用，因此在作物抗逆基因工程中具有较高潜在应用价值；AmNAC5可以显著提高抗旱性和耐盐性，并降低对外源ABA的敏感性。此外，AmNAC1~AmNAC5可以不同程度地提高转基因拟南芥细胞膜的稳定性、抗氧化胁迫能力和/或抗逆相关基因的转录水平，这可能是其发挥抗逆作用的重要生理与分子基础。.5. AmNAC4启动子的活性受多种非生物胁迫的诱导而增强。.这些研究结果不仅对于弄清AmNAC1~AmNAC5的抗逆功能和作用机理及其在作物抗逆基因工程中的利用价值提供了重要依据，而且为解析沙冬青和其他植物抗逆性的生理与分子机理提供了新的知识。